Hirudin Blood Research Articles

Assessment of platelet biology in equine patients with systemic inflammatory response syndrome.

In addition to maintaining hemostasis, platelets have an important role in modulating innate and adaptive immune responses. A low platelet count has been found to be a negative prognostic factor for survival in humans and horses with critical illnesses, such as sepsis or systemic inflammatory response syndrome (SIRS). Decreased platelet aggregation, caused by in vivo activation, has been found in human patients with severe sepsis. In our prospective controlled study, we assessed platelet biology in blood samples from 20 equine SIRS cases and 120 healthy control horses. Platelet variables such as platelet count, large platelet count, clumps, plateletcrit, mean platelet volume, and mean platelet component concentration were analyzed by laser flow cytometry (Advia 2120) from K3EDTA blood and from citrate blood. Hirudin blood samples were analyzed by impedance aggregometry (Multiplate analyzer; Roche) for platelet aggregation, including spontaneous aggregation and aggregation by 4 different agonists: adenosine diphosphate (ADPtest), ADP + prostaglandin E1 (ADPtestHS), arachidonic acid (ASPItest), and collagen (COLtest). SIRS cases had significantly lower platelet counts in K3EDTA blood (p < 0.0001) compared to control horses. There were no significant differences in aggregation values between SIRS cases and controls. Non-surviving SIRS horses did not have statistically significant lower platelet counts or lower aggregation values for COLtest, ADPtest, or ADPtestHS compared to surviving SIRS horses, although 5 non-survivors were thrombocytopenic.

Effects of Time-Interval since Blood Draw and of Anticoagulation on Platelet Testing (Count, Indices and Impedance Aggregometry): A Systematic Study with Blood from Healthy Volunteers.

Platelet count, indices (mean volume, young—immature platelet fraction) and aggregation are widely used laboratory parameters to investigate primary hemostasis. We performed a systematic, thorough evaluation of the influence of the time-interval since blood draw from 20 healthy individuals and of the anticoagulation of collected blood on such parameters. Blood was anticoagulated with citrate, K2-ethylenediaminetetraacetic acid (EDTA) and hirudin and analyzed 5, 30, 60, 120 and 180 min after blood draw. Multiple electrode aggregometry (MEA) was performed with either hirudin (half-diluted with NaCl) or citrate samples (half-diluted with NaCl or CaCl2 3 mM). Platelet count and indices (Sysmex XN-20) were rather stable over time with EDTA blood. MEA results were lower with citrate blood than with hirudin blood; supplementation with calcium was partially compensatory. MEA results were also lower when performed less than 30 or more than 120 min after blood draw. Platelet clumping, quantitatively estimated with microscope examination of blood smears, was more important in hirudin blood than citrate or EDTA blood and could explain some of the differences observed between preanalytical variables. The results stress once more the importance of preanalytical variables in hemostasis laboratory testing. Decision thresholds based on those tests are only applicable within specific preanalytical conditions.

Time dependent reduction in platelet aggregation using the multiplate analyser and hirudin blood due to platelet clumping

The Multiplate is a popular instrument that measures platelet function using whole blood. Potentially considered a point of care instrument, it is also used by hemostasis laboratories. The instrument is usually utilized to assess antiplatelet medication or as a screen of platelet function. According to the manufacturer, testing should be performed within 0.5–3 hours of blood collection, and preferably using manufacturer provided hirudin tubes. We report time-associated reduction in platelet aggregation using the Multiplate and hirudin blood collection tubes, for all the major employed agonists. Blood for Multiplate analysis was collected into manufacturer supplied hirudin tubes, and 21 consecutive samples assessed using manufacturer supplied agonists (ADP, arachidonic acid, TRAP, collagen and ristocetin), at several time-points post-sample collection within the recommended test time period. Blood was also collected into EDTA as a reference method for platelet counts, with samples collected into sodium citrate and hirudin used for comparative counts. All platelet agonists showed a diminution of response with time. Depending on the agonist, the reduction caused 5–20% and 22–47% of responses initially in the normal reference range to fall below the reference range at 120min and 180min, respectively. Considering any agonist, 35% and 67% of initially “normal” responses became ‘abnormal’ at 120 min and 180 min, respectively. Platelet counts showed generally minimal changes in EDTA blood, but were markedly reduced over time in both citrate and hirudin blood, with up to 40% and 60% reduction, respectively, at 240 min. The presence of platelet clumping (micro-aggregate formation) was also observed in a time dependent manner, especially for hirudin. In conclusion, considering any platelet agonist, around two-thirds of samples can, within the recommended 0.5–3 hour testing window post-blood collection, yield a reduction in platelet aggregation that may lead to a change in interpretation (i.e., normal to reduced). Thus, the stability of Multiplate testing can more realistically be considered as being between 30–120 min of blood collection for samples collected into hirudin.

Optimization of Multiplate® whole blood platelet aggregometry in the Beagle dog and Wistar rat for ex vivo drug toxicity testing

This study was performed to optimize and standardize the use of the Multiplate(®) whole blood impedance aggregometer in the Beagle dog and Wistar rat for use in a research laboratory environment. The anticoagulants citrate, heparin and hirudin were compared and platelet aggregation responses to ADP, collagen, arachidonic acid and Par-4 agonist were evaluated to determine their half maximal effective concentrations (EC(50)) in blood containing low concentrations of a drug solvent (0.1% DMSO). The results indicate that citrate anticoagulation is not suitable for Multiplate(®) whole blood aggregometry because of the presence of spontaneous aggregation. ADP and collagen were found to be appropriate agonists for both species, whereas in the Beagle dog Par-4 agonist failed to induce aggregation and arachidonic acid induced platelet aggregation showed a high interindividual variability. The agonists EC(50) calculated in hirudin blood were 2.70 μM ADP, 0.85 μg/ml collagen, 0.03 mM arachidonic acid and 165.7 μM Par-4 agonist in the Wistar rat, and 0.95 μM ADP and 0.23 μg/ml collagen in the Beagle dog.

Effect of different anticoagulants on multiple electrode platelet aggregometry after clopidogrel and aspirin administration in patients undergoing coronary stent implantation: A comparison between citrate and hirudin

Outcomes of platelet function tests are highly dependent on the type of blood anticoagulant used. The primary objective of this study was to clinically evaluate the platelet function after dual antiplatelet therapy using two different types of anticoagulant (citrate and hirudin). We compared data obtained from multiple electrode platelet aggregometry (MEA) with reference to light transmission aggregometry (LTA) and VerifyNow (VN) assays. Blood samples were obtained from 119 patients on dual antiplatelet therapy at the time of PCI (PCI) and the following morning (post-PCI). The platelet function tests were performed using two anticoagulated (citrate or hirudin) blood types for MEA as well as citrated blood for LTA and VerifyNow assays. ADP-induced MEA values at PCI for citrated and hirudinated anticoagulants were 36.5 ± 14.3 AUC and 41.4 ± 18.2 AUC (p = 0.021) and post-PCI values were 28.2 ± 11.9 AUC and 28.3 ± 12.8 AUC (p = 0.95). Additionally, AA-induced MEA values at PCI by citrated and hirudinated blood was 13.4 ± 7.3 AUC and 17.6 ± 13.4 AUC (p < 0.01). Post-PCI AA-induced MEA values were 12.0 ± 6.7 AUC and 13.5 ± 8.5 AUC (p = 0.12), respectively. Significant correlations were observed between the two anticoagulants used for MEA and LTA or VN values under ADP-induced platelet stimulation. Citrate tubes are clinically adequate for MEA assays and provide a more economical alternative to hirudin for early and/or delayed phases after clopidogrel-loading doses.

Evaluation of the INNOVANCE PFA P2Y test cartridge: Sensitivity to P2Y12 blockade and influence of anticoagulant

Monitoring of platelet ADP receptor P2Y12 inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100® cartridge formulations to detect P2Y12 inhibition, a new cartridge for the PFA-100 (INNOVANCE® PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow® P2Y12 assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10 µM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30 min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10 µM R-138727. Intermediate results were typically obtained with 1 µM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1 µM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N = 5) revealed that all platelet tests (except CEPI) became more sensitive to 1 µM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y12 inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in vitro inhibition of P2Y12 is markedly different depending on the anticoagulant used.

Monitoring of clopidogrel treatment by multiple electrode platelet aggregometry1,2

Abstract Low responsiveness to clopidogrel is an important cause of adverse events after coronary stenting. Multiple electrode platelet aggregometry (MEA) measured by the Multiplate® system seems to be suitable for monitoring clopidogrel treatment. We validated this method by analyzing hirudin blood samples from 150 healthy volunteers to establish reference ranges of the MEA tests, from 75 hematologic patients with anemia or thrombopenia to determine the dependence of MEA values on hematocrit (HCT) and platelet count, and from 1005 patients after percutaneous coronary intervention (PCI) and 600 mg clopidogrel loading dose to test the reproducibility of the results under clinical conditions. It was found that MEA results are not affected by the different methods of blood withdrawal and sample transport, if the blood is kept at rest for 30 min before measuring. Measuring must be performed within 3 h after blood withdrawal. It was found that MEA results are not valid in patients with HCT ≤0.30 L/L or platelets ≤100/nL. Because clopidogrel loading also causes a decrease of thrombin receptor-activating peptide (TRAP) values, clopidogrel responsiveness, defined by ADP values under the lower reference level (men: 32 U, women: 46 U), can only reliably be classified if the TRAP value is &gt;20 U and the TRAP/ADP ratio &lt;3. Clopidogrel monitoring should be performed within 24 h after PCI, despite the risk of unclear results in ∼12%. In summary, by concentrating on preanalytical conditions and blood count, MEA is suitable to detect clopidogrel response. The TRAP test should be performed simultaneously to allow a valid assessment.

Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement.

In patients undergoing percutaneous coronary intervention (PCI), a link between bleeding and excess mortality has been demonstrated. A potential association of platelet response to clopidogrel and bleeding has not been well established yet. The aim of the present study was to assess the impact of clopidogrel responsiveness on the risk of bleeding in clopidogrel-treated patients undergoing PCI. Patients (n=2533) undergoing PCI after pretreatment with 600 mg of clopidogrel were enrolled in this study. Blood was obtained directly before PCI. Adenosine-diphosphate (ADP)-induced platelet aggregation was assessed on a Multiplate analyzer. The primary endpoint was the incidence of in-hospital Thrombolysis in Myocardial Infarction (TIMI) major bleeding and the secondary endpoint was in-hospital TIMI minor bleeding. Receiver-operator curve (ROC) analysis was used to derive the optimal platelet aggregation value defining enhanced clopidogrel responders for the association of measurements with major bleeding. Thirty-four (1.3%) major bleeding events and 137 (5.4%) minor bleeding events were observed. The risk of a major bleeding was significantly higher in patients (n=975) with an enhanced response to clopidogrel as compared with the remaining patients (n=1558) (2.2 vs. 0.8%, unadjusted odds ratio (OR) 2.6, 95% confidence interval (CI) 1.3-5.2, P=0.005; adjusted OR 3.5, 95% CI 1.6-7.3, P=0.001). No significant differences between both groups were observed for the occurrence of minor bleeding events (P=0.68). Enhanced clopidogrel responsiveness is associated with a higher risk of major bleeding. Whether guidance of antiplatelet treatment based on platelet function testing proves useful for avoiding bleeding events warrants further investigation.

Prothrombin conversion intermediate effectively neutralizes toxic levels of hirudin

Meizothrombin, the stable intermediate product of ecarin-induced prothrombin conversion, was investigated for its ability to bind hirudin in blood. After in vitro pre-incubation of rat plasma with ecarin, the prolongation of the thrombin time caused by hirudin was reduced. The extent of hirudin neutralization was found to be dependent on the duration of incubation with ecarin. In vivo, after bilateral nephrectomy in Wistar rats and following administration of hirudin at a dose of 1 or 5 mg/kg, the blood level of hirudin remained constant after 2 h. After infusion of ecarin following hirudin administration, the hirudin blood level dropped sharply, reaching significantly reduced values, and bleeding stopped. Platelet count and fibrinogen level in plasma remained unchanged in the experiments using ecarin-induced prothrombin conversion intermediate generation. It is concluded that meizothrombin, a naturally occurring prothrombin conversion intermediate, provides an effective agent to neutralize toxic blood levels of hirudin.

The mechanism of the action of anti-pneumococcic serum

A year ago it was observed that an intravenous injection of a small amount (0.2 c.c.) of immune serum causes the disappearance, within ten minutes'time, of the bacteria from the blood of a rabbit having a pneumococcic septicaemia. It was decided to investigate the above phenomenon in the hope of ascertaining, if possible, the manner of action of anti-pneumococcic serum.In the light of our results concerning the behavior of typhoid bacilli in the circulation of normal rabbits, we believed it possible that whole rabbit blood plus immune serum killed the pneumococci. Therefore rabbit blood was taken in hirudin; varying amounts of immune serum were added to the hirudin blood; definite quantities of bacteria were added and plates made immediately after starting the experiments, and at intervals for twenty-four hours. The colonies were found much reduced in the tubes containing the immune serum and whole blood, but after twenty-four hours all tubes were “saturated” with bacteria. It was believed that the immune ...